[Williams syndrome: its clinical aspects and molecular bases]. / Síndrome de Williams: aspectos clínicos y bases moleculares.

INTRODUCTION AND DEVELOPMENT: Williams syndrome is a developmental disorder with an estimated prevalence of 1 in 7,500 newborns. Its phenotype is characterized by distinctive facial features, mild to moderate mental retardation and general cognitive deficits with a non-uniform profile, having problems in some areas (psychomotricity, visuospatial integration) and relative preservation of others (language, musicality), friendly personality, occasional hypercalcemia of infancy, and a vasculopathy with supravalvular aortic stenosis. Williams syndrome is caused by a submicroscopic deletion of 1.55 Mb in the chromosome band 7q11.23, which includes 26-28 genes. The mutational mechanism consists in a misalignment between regions of almost identical sequence and the subsequent unequal recombination. The reciprocal product of this rearrangement is the duplication of this region, causing a language specific disorder. CONCLUSIONS: Clinical-molecular correlations establishment through a good phenotypic characterization and the precise analysis of breakpoints in patients with atypical and typical deletions, altogether with the design of animal models and functional studies in vitro for the genes of the interval will be important to be able to determine the exact contribution of the genes to the phenotype, to know their pathogenesis and physiopathology, and to identify therapeutic methods.

Frataxin point mutations in two patients with Friedreich's ataxia and unusual clinical features.

Two patients with a progressive ataxia are presented with clinical features consistent with classic Friedreich's ataxia (FRDA), but also with features unusual for FRDA. Analysis of DNA showed that each patient is heterozygous for the expanded GAA repeat of FRDA, but carries a base change on his other frataxin allele. For one patient a non-conservative arginine to cysteine amino acid change is predicted at amino acid 165 whereas the other mutation is found at the junction of exon one and intron one. Muscle biopsy showed an absence of frataxin immunoreactivity in the patient harbouring the intronic mutation, confirming the pathological nature of the base change. These mutations extend the range of point mutations seen in FRDA, and agree with recent reports suggesting phenotypic variation in patients with FRDA harbouring point mutations in conjunction with an expanded GAA repeat.

Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase.

Frataxin is a mitochondrial protein deficient in Friedreich ataxia (FRDA) and which is associated with abnormal intramitochondrial iron handling. We identified the mitochondrial processing peptidase beta (MPPbeta) as a frataxin protein partner using the yeast two-hybrid assay. In in vitro assays, MPPbeta binds frataxin which is cleaved by the reconstituted MPP heterodimer. MPP cleavage of frataxin results in an intermediate form (amino acids 41-210) that is processed further to the mature form. In vitro and in vivo experiments suggest that two C-terminal missense mutations found in FRDA patients modulate interaction with MPPbeta, resulting in a slower maturation process at the normal cleavage site. The slower processing rate of frataxin carrying such missense mutations may therefore contribute to frataxin deficiency, in addition to an impairment of its function.

Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes.

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.

Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.

Friedreich's ataxia is due to loss of function mutations in the gene encoding frataxin (FRDA). Frataxin is a protein of unknown function. In situ hybridization analyses revealed that mouse frataxin expression correlates well with the main site of neurodegeneration, but the expression pattern is broader than expected from the pathology of the disease. Frataxin mRNA is predominantly expressed in tissues with a high metabolic rate, including liver, kidney, brown fat and heart. We found that mouse and yeast frataxin homologues contain a potential mitochondrial targeting sequence in their N-terminal domains and that disruption of the yeast gene results in mitochondrial dysfunction. Finally, tagging experiments demonstrate that human frataxin co-localizes with a mitochondrial protein. Friedreich's ataxia is therefore a mitochondrial disease caused by a mutation in the nuclear genome.

Evolution of the Friedreich's ataxia trinucleotide repeat expansion: founder effect and premutations.

Friedreich's ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for approximately 17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through "premutation" intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.

Clinical and genetic abnormalities in patients with Friedreich's ataxia.

BACKGROUND: Friedreich's ataxia, the most common inherited ataxia, is associated with a mutation that consists of an unstable expansion of GAA repeats in the first intron of the frataxin gene on chromosome 9, which encodes a protein of unknown function. METHODS: We studied 187 patients with autosomal recessive ataxia, determined the size of the GAA expansions, and analyzed the clinical manifestations in relation to the number of GAA repeats and the duration of disease. RESULTS: One hundred forty of the 187 patients, with ages at onset ranging from 2 to 51 years, were homozygous for a GAA expansion that had 120 to 1700 repeats of the trinucleotides. About one quarter of the patients, despite being homozygous, had atypical Friedreich's ataxia; they were older at presentation and had intact tendon reflexes. Larger GAA expansions correlated with earlier age at onset and shorter times to loss of ambulation. The size of the GAA expansions (and particularly that of the smaller of each pair) was associated with the frequency of cardiomyopathy and loss of reflexes in the upper limbs. The GAA repeats were unstable during transmission. CONCLUSIONS: The clinical spectrum of Friedreich's ataxia is broader than previously recognized, and the direct molecular test for the GAA expansion on chromosome 9 is useful for diagnosis, determination of prognosis, and genetic counseling.

Blue-light receptor requirement for gravitropism, autochemotropism and ethylene response in Phycomyces.

Light, gravity and ethylene represent for plants and fungi important environment cues for spatial orientation and growth regulation. Coordination of the frequently conflicting stimuli requires signal-integration sites, which, however, remain largely unidentified. The genetic and physiological basis for signal integration was investigated with a set of phototropism mutants (genotype mad) of the UV- and blue-light-sensitive fungus Phycomyces blakesleeanus, which responds also to gravity, ethylene and nearby obstacles (autochemotropism or avoidance response). Both, class 1 and class 2 mutants display a reduced sensitivity to visible light. Class 1 mutants with defects in genes madA, B, C, I have preserved their sensitivity to gravity and ethylene, whereas class 2 mutants with defects in genes madD,E,F,G,J have lost it. We found that the phototropic sensitivity of class 1 mutants is affected roughly to the same extent in far UV and blue light. In contrast, the sensitivity loss of class 2 mutants is restricted mainly to the near-UV and the blue-light region, whereas the sensitivity to far UV is only mildly affected. This behavior of the class 2 mutants indicates that different photoreceptors mediate phototropism in far-UV and in near-UV/ blue light. The photogravitropic action spectra for two class 2 mutants with defects in genes madF and madJ display distortions between 342 and 530 nm and a bathochromic shift relative to the action spectrum of the wild type. These features indicate that the madF and madJ mutants are affected at the level of the blue-light photoreceptor system. As an implication we infer that an intact near-UV/blue-light photoreceptor system is required even in darkness for negative gravitropism, the ethylene response and autochemotropism. In Phycomyces, signal integration occurs, at least in part, at the level of the near-UV/blue-light photoreceptor system.

Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

The Friedreich ataxia critical region spans a 150-kb interval on chromosome 9q13.

By analysis of crossovers in key recombinant families and by homozygosity analysis of inbred families, the Friedreich ataxia (FRDA) locus was localized in a 300-kb interval between the X104 gene and the microsatellite marker FR8 (D9S888). By homology searches of the sequence databases, we identified X104 as the human tight junction protein ZO-2 gene. We generated a large-scale physical map of the FRDA region by pulsed-field gel electrophoresis analysis of genomic DNA and of three YAC clones derived from different libraries, and we constructed an uninterrupted cosmid contig spanning the FRDA locus. The cAMP-dependent protein kinase gamma-catalytic subunit gene was identified within the critical FRDA interval, but it was excluded as candidate because of its biological properties and because of lack of mutations in FRDA patients. Six new polymorphic markers were isolated between FR2 (D9S886) and FR8 (D9S888), which were used for homozygosity analysis in a family in which parents of an affected child are distantly related. An ancient recombination involving the centromeric FRDA flanking markers had been previously demonstrated in this family. Homozygosity analysis indicated that the FRDA gene is localized in the telomeric 150 kb of the FR2-FR8 interval.

Isolation, characterization and transformation, by autonomous replication, of Mucor circinelloides OMPdecase-deficient mutants.

Pyrimidine auxotrophs of Mucor circinelloides were isolated after mutagenesis with nitrosoguanidine and selected for resistance to 5-fluoroorotate. These mutants were genetically and biochemically characterized and found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase) activity or in orotate phosphoribosyl transferase (OPRTase) activity. Different circular DNA molecules containing the homologous pyrG gene were used to transform a representative OMPdecase-deficient strain to uracil prototrophy. Southern analysis, as well as mitotic stability analysis of the transformants, showed that the transforming DNA is always maintained extrachromosomally. The smallest fragment tested that retained both the capacity to complement the pyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kb M. circinelloides genomic DNA fragment. This fragment consists of the pyrG coding region flanked by 606 nucleotides at the 5' and 330 nucleotides at the 3' ends, respectively. Sequence analysis reveals that it does not share any element in common with another M. circinelloides genomic DNA fragment which also promotes autonomous replication in this organism, except those related to transcription. Furthermore, it differs from elements which have been shown to be involved in autonomous replication in other fungal systems. An equivalent plasmid harbouring the heterologous Phycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication in M. circinelloides may be driven by elements normally present in nuclear coding genes.

Genetic characterization of two phototropism mutants of Phycomyces with defects in the genes madI and madJ.

Two Phycomyces genes, madI and madJ, which are involved in phototropism, were characterized by recombination and complementation analyses. The madI gene was located on linkage group IV of the genetic map of Phycomyces, 27 map units away from the gene carA. Complementation and recombination studies involving the genes madD, madE, madF, and madG, in combination with previous genetic studies, show that the recently isolated mad-407 mutation defines a novel behavioural gene, madJ, of Phycomyces. A regulatory role of the madJ gene product in the light-sensory transduction pathway is suggested.

Isolation and characterization of phototropism mutants of Phycomyces insensitive to ultraviolet light.

Phototropism mutants of the zygomycete fungus Phycomyces blakesleeanus were isolated on the basis of their loss of responsivity to UV light. Four of these mutants had retained a partial sensitivity to near-UV and to blue light. Gravitropism and the avoidance response were unaffected in these mutants. One mutant, A909, had lost most of its sensitivity to near-UV and blue light while the sensitivity to far-UV light was only slightly affected. Additionally, the gravitropic and the avoidance responses were significantly reduced in A909. A complementation analysis of the five strains of Phycomyces with known phototropism mutants indicated that strains A896, A897, and A898 were defective in the madA gene, and that A905 was affected in the madC gene. In strain A909 the input, as well as the output, of the transduction chain is affected.

Isolation, characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus.

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5'-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.

Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase: use of pyrG for homologous transformation.

A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.

A new gene (madI) involved in the phototropic response of Phycomyces.

Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.